Biology subjects

Bullen, C. K.

Publications and source records attributed to Bullen, C. K..

5 recordsLinked to original sources

bioRxiv2025

APOE genotype confers context dependent neurovascular vulnerability in immune-vascularized human forebrain organoids

The APOE gene is a major genetic determinant of neurovascular and immune function, yet the mechanisms by which its isoforms modulate brain vulnerability to pathogenic stress remain incompletely understood. Here, we employ isogenic human iPSC-derived immune-vascularized--Forebrain Organoid-based Multicellular Assembled Cerebral Organoids (FORMA-COs)--to dissect isoform-specific responses to a clinically relevant viral challenge. We find that APOE2/2 and APOE4/4 FORMA-COs exhibit heightened viral RNA burden and distinct neuroinflammatory profiles compared to APOE3/3. Specifically, APOE4/4 promotes IL-1 and VEGFA induction, whereas APOE2/2 leads to elevated TNF-{beta} and VEGFA protein accumulation, indicating divergent pathways of injury. Integrated transcriptomic analyses, combined with known and predicted APOE protein-protein interaction networks, reveal genotype-dependent enrichment of cytokine signaling, angiogenic remodeling, and immune dysregulation. In vivo validation using humanized mouse models corroborates APOE genotype- specific vascular remodeling, microglial activation, and oligodendrocyte perturbation. These findings demonstrate that APOE genotype confers context-specific susceptibility to neuroimmune and vascular injury, providing insight into genetic risk mechanisms underlying infection-related and neurodegenerative brain disorders.

neuroscience↗

bioRxiv2022

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guerin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

immunology↗

bioRxiv2021

Allosteric cooperation in β-lactam binding to a non-classical transpeptidase

Mycobacterium tuberculosis peptidoglycan (PG) is atypical as its synthesis involves a new enzyme class, L,D-transpeptidases. Prior studies of L,D-transpeptidases have identified only the catalytic site that binds to peptide moiety of the PG substrate or {beta}-lactam antibiotics. This insight was leveraged to develop mechanism of its activity and inhibition by {beta}-lactams. Here we report identification of an allosteric site at a distance of 21 [A] from the catalytic site that binds the sugar moiety of PG substrates (hereafter referred to as the S-pocket). This site also binds a second {beta}-lactam molecule and influences binding at the catalytic site. We provide evidence that two {beta}-lactam molecules bind co-operatively to this enzyme, one non-covalently at the S-site and one covalently at the catalytic site. This dual {beta}-lactam binding phenomenon is previously unknown and is an observation that may offer novel approaches for the structure-based design of new {beta}-lactam antibiotics for M. tuberculosis.

biochemistry↗

bioRxiv2021

Sulforaphane exhibits in vitro and in vivo antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are no orally available medications for prophylaxis for those exposed to SARS-CoV-2 and limited therapeutic options for those who develop COVID-19. We evaluated the antiviral activity of sulforaphane (SFN), a naturally occurring, orally available, well-tolerated, nutritional supplement present in high concentrations in cruciferous vegetables with limited side effects. SFN inhibited in vitro replication of four strains of SARS-CoV-2 as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN is a promising treatment for prevention of coronavirus infection or treatment of early disease.

microbiology↗

bioRxiv2018

A second generation IL-2 receptor-targeted diphtheria fusion toxin exhibits anti-tumor activity and synergy with anti-PD-1 in melanoma

Denileukin diftitox (DAB1-389-IL-2, Ontak(R)) is a diphtheria toxin-based fusion protein that depletes CD25-positive cells including regulatory T cells (Tregs) and was approved for the treatment of persistent or recurrent cutaneous T cell lymphoma. However, the clinical use of denileukin diftitox was limited by vascular leak toxicity and production issues related to drug aggregation and purity. We found that a single amino acid substitution (V6A) in a motif associated with vascular leak induction yields a fully active, second-generation biologic, s-DAB1-386-IL-2(V6A), which elicits 50-fold less HUVEC monolayer permeation and is 3.7-fold less lethal to mice by LD50 analysis than s-DAB1-386-IL-2 Additionally, to overcome aggregation problems, we developed a novel production method for the fusion toxin using Corynebacterium diphtheriae that secretes fully-folded, biologically active, monomeric s-DAB1-386-IL-2 into the culture medium. Using the poorly immunogenic mouse B16F10 melanoma model, we initiated treatment 7 days after tumor challenge and observed that, while both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 are inhibitors of tumor growth, the capacity to treat with higher doses of s-DAB1-386-IL-2(V6A) could provide a superior activity window. In a sequential dual therapy study in tumors that have progressed for 10 days both s-DAB1-386-IL-2(V6A) and s-DAB1-386-IL-2 given prior to checkpoint inhibition with anti-PD-1 antibodies inhibited tumor growth, while either drug given as monotherapy had less effect. s-DAB1-386-IL-2(V6A), a fully monomeric protein with reduced vascular leak, is a second-generation diphtheria toxin-based fusion protein with promise as a cancer immunotherapeutic both alone and in conjunction with PD-1 blockade.\n\nSignificance StatementRegulatory T cells (Tregs) infiltrate tumors in various cancers and promote an immunosuppressive microenvironment that hinders anti-tumor immunity. Denileukin diftitox, a diphtheria toxin-based fusion protein that depletes Tregs, was approved for the treatment of T cell malignancies, but its clinical use was limited due to the presence of protein aggregates and toxicity associated with vascular leakage. Here we report the production of a second generation IL-2 receptor-targeted, fully-folded, monomeric diphtheria fusion toxin, and a V6A mutant variant which showed reduced vascular leak in vitro and reduced lethality in mice. In a mouse model of melanoma, we found significant decrease in tumor growth associated with reduction in Tregs when the protein was tested as monotherapy or in combination with checkpoint blockade.

cancer biology↗